The broad objective of this research project is to explore the mechanisms which regulate the level of expression of specific membrane proteins and their spatial distribution in nerve and muscle. The focus of current research is upon the sodium- and potassium-ion stimulated ATPase. Our exploration of Na/K ATPase regulation is subdivided into six projects. First, the genetic basis of the Na/K ATPase is being described through cloning and sequencing the genes and gene transcripts encoding each of the subunits. Second, the expression of each gene in various cell types of nerve and muscle is being described through use of isoform-specific DNA probes and isoform-specific monoclonal antibodies. Third, up- and down-regulation of the Na/K ATPase in skeletal muscle is being examined in detail at both nucleic acid and protein levels. In this study, four mechanisms of regulation have been identified: these occur at the level of transcription, subunit assembly, incorporation into plasma membrane, and protein turnover. The underlying mechanisms are being investigated. Fourth, a number of structure/function and structure/bioregulation relationships are being examined through expression of avian Na/K ATPase subunits in mouse L cell and myogenic cell lines, including studies on the nature of the cardiac glycoside binding site, the requirements for subunit assembly, and targeting to plasma membrane. Fifth, plasma membrane dynamics during nerve growth are being studied in tissue cultured sensory neurons, with focus upon determining the major sites of insertion of newly synthesized Na/K ATPase molecules in relation to the growth cone and extending axons. Sixth, the Na/K ATPase of Drosophila is being studied with particular attention to roles of the Na/K ATPase in early development and possible behavioral consequences of Na/K ATPase mutations.
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